Decoding how a soil bacterium extracts building blocks and metabolic energy from ligninolysis provides road map for lignin valorization

Abstract

Sphingobium sp. SYK-6 is a soil bacterium boasting a well-studied ligninolytic pathway and the potential for development into a microbial chassis for lignin valorization. An improved understanding of its metabolism will help researchers in the engineering of SYK-6 for the production of value-added chemicals through lignin valorization. We used ¹³C-fingerprinting, ¹³C metabolic flux analysis (¹³C-MFA), and RNA-sequencing differential expression analysis to uncover the following metabolic traits: (i) SYK-6 prefers alkaline conditions, making it an efficient host for the consolidated bioprocessing of lignin, and it also lacks the ability to metabolize sugars or organic acids; (ii) the CO₂ release (i.e., carbon loss) from the ligninolysis-based metabolism of SYK-6 is significantly greater than the CO₂ release from the sugar-based metabolism of Escherichia coli; (iii) the vanillin catabolic pathway (which is the converging point of majority of the lignin catabolic pathways) is coupled with the tetrahydrofolate-dependent C1 pathway that is essential for the biosynthesis of serine, histidine, and methionine; (iv) catabolic end products of lignin (pyruvate and oxaloacetate) must enter the tricarboxylic acid (TCA) cycle first and then use phosphoenolpyruvate carboxykinase to initiate gluconeogenesis; and (v) ¹³C-MFA together with RNA-sequencing differential expression analysis establishes the vanillin catabolic pathway as the major contributor of NAD(P)H synthesis. Therefore, the vanillin catabolic pathway is essential for SYK-6 to obtain sufficient reducing equivalents for its healthy growth; cosubstrate experiments support this finding. This unique energy feature of SYK-6 is particularly interesting because most heterotrophs rely on the transhydrogenase, the TCA cycle, and the oxidative pentose phosphate pathway to obtain NADPH.

Keywords: 13C-MFA; NADPH; fingerprinting; gluconeogenesis; lignin.

Fig. 1.

Vanillin and vanillic acid are the monomers generated from the enzymatic cleavage of some of the major linkages present in lignin. Vanillin and vanillic acid undergo further chemical transformation through a linear set of reactions before cleaving into pyruvate and oxaloacetate. Pyruvate and oxaloacetate then enter the central metabolic pathway of SYK-6 for the generation of its cellular building blocks and energy. SW, softwood.

Fig. 2.

Incorporation of ¹³C-carbon into the amino acids ([M-57]⁺) of SYK-6 after feeding with the following labeled carbon substrates: (A) (1,2-¹³C) glucose and yeast extract; (B) (phenyl-¹³C) vanillin; (C) (1,2-¹³C) acetate; and (D) (U-¹³C) pyruvate*. The asterisk indicates that unlabeled glyoxylate was added to the labeled pyruvate culture to explore the presence of glyoxylate shunt in SYK-6. The SD represents the 2% technical error of the instrument. The Embden–Meyerhof–Parnas pathway is shown in purple; the TCA is shown in green; the PPP is shown in red; and the amino acid synthesis pathway is shown in black.

Fig. 3.

Growth of SYK-6 under varying pH conditions. (A) With vanillic acid plus pyruvate as the carbon source. (B) With vanillin plus pyruvate as the carbon source.

Fig. 4.

C1 metabolism in SYK-6. (A) The THF-dependent one-carbon metabolic pathway in SYK-6 for the synthesis of methionine, serine, and AMP. The demethylation step is shown for vanillic acid as an example. The carbon derived from the methyl group is shown in blue in each molecule to allow the tracking of this carbon in the formation of amino acids. The gene symbols in the figure are based on KEGG annotations. A detailed pathway for the synthesis of AMP is presented in SI Appendix, Fig. S1. (B) Pathway for the biosynthesis of histidine in SYK-6. Carbon from the C1-THF pathway enters into the histidine backbone through adenosine triphosphate that was synthesized from N¹⁰-formyl THF. The remaining five carbons for histidine synthesis come from R5P, an intermediate of the PPP. ATP is shown in blue to track the carbons that take part in histidine synthesis. The gene symbols in the figure are based on KEGG annotations. F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; P, phosphite (PO₃) ion; R5P, ribose-5-phosphate.

Fig. 5.

Alanine labeling reveals the activity of cataplerotic pathways in SYK-6. The MID shown was obtained from SYK-6 cells fed with 1,2-¹³C acetate.

Fig. 6.

Metabolic flux distribution of Sphingobium sp. SYK-6 growing on vanillin in W medium. The flux values are all relative to the vanillin uptake rate. 3PG, 3-phosphoglycerate; 6PG, 6-phosphogluconate; AceCoA, acetyl-CoA; AKG, α-ketoglutarate; CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde; CIT, citrate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; FUM, fumarate; G6P, glucose 6-phosphate; GAP, glyceraldehyde 3-phosphate; GLX, glyoxylate; ICT, isocitrate; MAL, malate; OAA, oxaloacetate; PDC, 2-pyrone-4,6-dicarboxylate; PEP, phosphoenolpyruvate; PYR, pyruvate; R5P, ribose 5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; SER, serine; SUC, succinate; SucCoA, succinyl-CoA; X5P, xylulose-5-phosphate.

Fig. 7.

Heat map of differentially expressed genes involved in NAD(P)H metabolism of Sphingobium SYK-6 based on RNA-seq data analysis. The fold changes are reported for cells grown in the presence of vanillin (a monomeric product of lignin) or GGE (a dimeric product of lignin) vs. cells grown in the presence of pyruvate (a carbon source lacking the vanillin catabolic pathway). A plus or minus sign indicates that the corresponding gene is involved in the production or consumption of NAD(P)H, respectively. The results are shown in triplicate for each carbon source, and each column represents the results from one of the biological replicates. 3PG, 3-phosphoglycerate; 6PGL, 6-phosphogluconolactone; CHMS, 4-carboxy-2-hydroxymuconate-6-semialdehyde; PDC, 2-pyrone-4,6-dicarboxylate; AceCoA, acetyl-CoA; AKG, α-ketoglutarate; G6P, glucose 6-phosphate; GAP, glyceraldehyde 3-phosphate; GGE, Guaiacylglycerol-β-guaiacyl ether; ICIT, isocitrate; MAL, malate; MPHPV, α-(2-methoxyphenoxy)-β-hydroxypropiovanillone; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; SucCoA, succinyl-CoA.

Fig. 8.

Growth of SYK-6 in the presence of multiple substrates. This experiment was designed to verify the cosubstrate metabolism of SYK-6. However, the addition of other carbon intermediates to cultures containing pyruvate did not improve the growth of SYK-6. This experiment supports the need for the vanillin catabolic pathway to provide the extra NAD(P)H to boost the growth of SYK-6. Asp, aspartate; Form, formate; OAA, oxaloacetate; Pyr, pyruvate.